The diversity of methanogen-specific methyl-coenzyme M reductase alpha-subunit (mcrA/mrtA) genes in Italian rice field soil was analysed using a combination of molecular techniques and enrichment cultures. From 75 mcrA/mrtA clones retrieved from rice field soil, 52 were related to members of the Methanosarcinaceae, Methanosaetaceae and Methanobacteriaceae. However, 19 and four clones formed two novel clusters of deeply branching mcrA sequences, respectively, which could not be affiliated to known methanogens. A new methanogen-specific fingerprinting assay based on terminal restriction fragment length polymorphism (T-RFLP) analysis of fluorescently labelled polymerase chain reaction (PCR) products allowed us to distinguish all environmental mcrA/mrtA sequences via group-specific Sau96I restriction sites. Even genes for the isoenzyme methyl-coenzyme M reductase two (mrtA) of Methanobacteriaceae present in rice field soil were represented by a unique 470 bp terminal restriction fragment (T-RF). Both cloning and T-RFLP analysis indicated a significant representation of novel environmental mcrA sequences in rice field soil (238 bp T-RF). To identify these mcrA sequences, methanogenic enrichment cultures with rice field soil as inoculum were established with H2/CO2 as substrates at a temperature of 50 degrees C, and these were monitored using molecular tools. In subsequent transfers of these enrichment cultures, cloning and T-RFLP analysis detected predominantly SSU rRNA genes of rice cluster I (RC-I), an uncultivated euryarchaeotal lineage discovered previously in anoxic rice field soil. In parallel, both mcrA cloning and T-RFLP analyses of the enrichment culture identified the more frequent cluster of novel environmental mcrA sequences as belonging to members of RC-I. Thus, we could demonstrate the genotype and phenotype of RC-I Archaea by the presence of a catabolic gene in a methanogenic enrichment culture before the isolation of pure cultures.